Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder in which preferential loss of motor neurons (MNs) results in paralysis and death. Although ALS is largely a sporadic disease, research has focused on heritable forms of the disorder because clinical and pathological evidence suggests common pathogenic mechanisms. Mutations in the gene FUS cause some of the most aggressive early-onset forms of ALS. In a recent study, our lab demonstrated in a mouse model of disease that mutant FUS causes motor neuron degeneration not by a loss-of-function, by a toxic gain-of-function that does not involve an excess of FUS activity. FUS is one of a number of RNA binding proteins ? including TDP-43 and hnRNP A1 ? that have been causally related to ALS. Our recent work ? together with related studies from several labs ? has led to a disease model in which the low complexity (LC) ?prion-like? domain of FUS and related proteins drives its phase transition to an irreversible, neurotoxic assembly. Our data demonstrates that ALS-related mutations in FUS increase the natural tendency of the protein to form these toxic assemblies, which trap and sequester other ribonucleoprotein granule components, including proteins involved in translational control. In this project we will explore the mechanisms of FUS toxicity in a series of transgenic and knock-in mutant mice with which we have modelled key aspect of the FUS-ALS phenotype. In addition, in vitro studies using motor neurons and astrocytes derived from these mouse models will be used to investigate cell autonomous and non-autonomous mechanisms of disease. In Aim 1, we will use a conditional knock-in mouse model to express mutant FUS in MNs or astrocytes, or more broadly in the nervous system to explore the effects of regulated mutant FUS expression on MN survival and function, and on gene expression changes that may underlie MN degeneration in FUS-ALS. We will also analyze mice with ALS-causing mutations in the LC domain of FUS to test the role of this critical domain in the disease. In Aim 2, we will use microfluidics to isolate MN axons and test the idea that FUS-dependent defects in axonal protein synthesis contribute to MN degeneration, and we will pursue our finding that hnRNP U selectively interacts with ALS-mutant FUS by exploring in vivo the functional role of this RNA binding protein in disease progression. Finally in Aim 3, we will apply a combination of single-cell RNA sequencing and topological data analysis to a mixed distribution of in vitro differentiated MNs derived from our FUS knock-in mutant mice. This sophisticated integration of in vivo and in vitro experimental systems, combined with our integrative computational and analytical approach will allow us to elucidate pathways of disease in vulnerable subpopulations of MNs and to identify potential therapeutic targets for the treatment of FUS-ALS and related forms of motor neuron disease.